The present invention relates to a method of dynamically culturing human embryonic stem cells (hESCs) which method can be utilized for large scale production of embryoid bodies (EBs) and EBs-derived-cells and cell lines and thus is particularly suitable for the generation of cell cultures utilized in cell replacement therapy.
Human embryonic stem cells (hESCs) are proliferative, undifferentiated stem cells capable of being maintained in an undifferentiated state while preserving their pluripotent capacity.
ESCs can differentiate into cell types having a particular, specialized function (i.e., “fully differentiated” cells) or to cells capable of being maintained in an undifferentiated state (i.e., “pluripotent stem cells”). Upon removal from their cultures, ESCs can spontaneously differentiate into three-dimensional cell aggregates, which, overtime, increase in cell number and complexity and form embryoid bodies (EBs) (Thomson et al., 1998). EBs are morphological structures comprised of a population of ESCs and/or embryonic germ cells (EGCs) which have undergone differentiation. EBs formation initiates following the removal of differentiation blocking factors from ES cell cultures. In the first step of EBs formation, ESCs proliferate into small masses of cells which then proceed with differentiation. In the first phase of differentiation, following 1-4 days in culture for human ESCs, a layer of endodermal cells is formed on the outer layer of the small mass, resulting in “simple EBs”. In the second phase, following 3-20 days post-differentiation, “complex EBs” are formed. Complex EBs are characterized by extensive differentiation of ectodermal and mesodermal cells and derivative tissues.
Stem-cell-derived-differentiated cells of specific lineages are of increasing importance for various therapeutic and tissue engineering applications. However, for both tissue regeneration and cell-replacement applications there is a need to develop methods of efficiently producing large quantities of EBs-derived-differentiated cells.
Prior art methods of generating EBs involve the initial aggregation of ESCs into spheroid, three-dimensional structures. Thus, when undifferentiated ESCs are removed from their feeder layers and transferred to liquid media using non-adherent tissue culture plates large aggregates of EBs are formed (Itskovitz-Eldor et al., 2000). However, the extent of EB aggregation should be carefully monitored and controlled since large agglomerated EBs are often accompanied with extensive cell death and necrosis due to mass transport limitations (Dang et al., 2002).
To overcome these limitations, methods of controlled agglomeration of EBs have been developed. These include the hanging drop method in which the ESCs are aggregated in hanging drops for two days prior to their transfer to liquid cultures. Another method utilizes semi-solid methylcellulose cultures in order to control EB's agglomeration (as disclosed by Dang et al., 2002 and in U.S. Pat. Appl. No. 20030119107). However, although adequate for small-scale laboratory purposes these systems are not amenable to large-scale clinical production due to the inability to control the extracellular components, which may affect EB's purity and reproducibility, as well as the cell types which can be readily generated therefrom [Maltepe, E. et al., (1997). Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature, 386: 403-7; Soria, B. (2001). In vitro differentiation of pancreatic beta-cells. Differentiation 68: 205-19; Zandstra, P. W. and Nagy, A. (2001). Stem cell bioengineering. Annu Rev Biomed Eng 3:275-305]. On the other hand, prior art attempts to culture EBs in spinner flasks, as a simple, large-scale process, resulted in either the formation of large cell-clumps within a few days (Wartenberg et al., 2001), or in a massive hydrodynamic damage to the cells when extensive mixing was used (Chisti, 2001).
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of dynamically generating hEBs devoid of the above described limitations.